Pharmacological characterization of cannabinoid receptor activity in the rat-isolated ileum myenteric plexus-longitudinal muscle preparation

Background and purpose:

Cannabinoid effects on intestinal transit are commonly evaluated in rats. We characterized the cannabinoid receptors mediating the inhibitory effect of 5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)-cyclohexyl]-phenol (CP 55,940), (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2), arachidonylethanolamide (AEA) and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) on contractions of the rat ileum myenteric plexus-longitudinal muscle (MPLM) preparation.

Experimental approach:

The interaction of each agonist was examined with the CB₁ and CB₂ receptor antagonist rimonabant and SR 144,528 respectively, on contractions elicited by electrical field stimulation (EFS) or exogenous ACh. The interaction of AEA with capsazepine, a TRPV₁ receptor antagonist, was also investigated.

Key results:

EFS with single and trains of pulses evoked neurogenic ACh-mediated twitch and rebound contractions respectively. The rank order of potency for inhibition was CP 55,940 = WIN 55,212-2 > AEA > Δ⁹-THC and AEA > WIN 55,212-2 =Δ⁹-THC = CP 55,940 respectively. The stereoisomer WIN 55,212-3 was without effect. Rimonabant antagonized the inhibition of the twitches with pK_B values of around 8.60, but only antagonized rebound contractions induced by WIN 55,212-2, AEA and Δ⁹-THC, with pA₂ values of around 6.80. Rimonabant increased the twitches but inhibited the rebound contractions. Contractions to exogenous ACh were not altered. These observations extended to the guinea pig ileum MPLM.

Conclusions and implications:

The rat MPLM contains CB₁ receptors and at least two non-CB₁-non-CB₂-non-TRPV₁ receptors attenuating EFS-evoked ACh-mediated contractions in an EFS frequency-dependent pre-synaptic and stereo-specific manner. Augmentation of the twitches by rimonabant may be through antagonism of an endocannabinoid tone or inverse agonism, whereas inhibition of the rebound contractions involved partial agonism.     

Factors influencing the release of acetylcholine from the myenteric plexus of the ileum of the guinea-pig and rabbit.

1 The effects of electrical stimulation, changes in external ion concentrations and various drugs on acetylcholine release from the myenteric plexus were measured by bioassay in the presence of physostigmine and by recording the responses of the longitudinal muscle. In preparations from the guinea-pig, the acetylcholine output per pulse increased with decreasing frequency of stimulation and reached its maximum at a frequency of 0.017 Hz (1/min) and thus ensured that the output per unit of time was constant at frequencies below 0.5 Hz. Spontaneous release was suppressed during stimulation at 0.017 Hz. 2 In the rabbit, the fractional acetylcholine release was lower than in the guinea-pig. The output per pulse increased with decreasing frequency of stimulation but at a lesser rate, with the effect that the output per unit decreased between 0.5 and 0.017 Hz. 3 In the guinea-pig, reduction of the Ca2+ concentration, addition to the bath fluid of Mn2+, ganglion-blocking drugs, morphine and catecholamines reduced output more at low than at high frequencies of stimulation. In the rabbit, acetylcholine output was less sensitive to changes in Ca2+ concentration and insensitive to Mn2+ and morphine. 4 In the guinea-pig, morphine and catecholamines depressed both the contractile response and acetylcholine output whereas Mn2+ in concentrations up to 125 muM, bretylium and ganglion-blocking drugs depressed only acetylcholine output. 5 In preparations from the guinea-pig, drugs blocking noradrenergic neurons or alpha-adrenoceptors, e.g. bretylium, phenoxybenzamine, thymoxamine and phentolamine, increased acetylcholine output during stimulation at high (1.5 to 10 Hz) but not at low frequencies. 6 The implications of these findings for the release of acetylcholine from different pools in the heterogeneous myenteric plexus are considered. The possible errors, introduced by the effects of physostigmine, on the size of the acetylcholine pools and on the transmission of impulses within the myenteric plexus are discussed.     

The inhibitory effect of neurotensin on the motor activity of rabbit ileum: dependence on the ionic environment

The possible ionic mechanisms underlying the inhibitory effect of neurotensin on the spontaneous phasic contractions of the longitudinal muscle of the isolated rabbit ileum were studied by varying the ionic environment. Neurotensin (0.06-60 nM) reduced and abolished the phasic contractions in a concentration-dependent manner. The effect of neurotensin was transient, and the phasic contractions began to recover after 1-3 min although neurotensin was not washed out. The response to neurotensin was very sensitive to alterations in the ionic composition of the bath medium. The changes in the effect of neurotensin brought about by changes in [Na+], [K+], [Ca2+], and [Cl-] suggest that neurotensin hyperpolarizes the smooth muscle. In addition, the effect of combined changes in [Ca2+] and [Cl-] points to a specific role of these ions in the action of neurotensin.     

Pharmacological evidence for the existence of interactions between dopaminergic and opioid peptidergic systems in guinea-pig ileum myenteric plexus

Apomorphine or bromocriptine treatment at doses that act on dopamine autoreceptors resulted in a significant elevation of the release of opioid peptides from the myenteric plexus of guinea-pig, since these drugs produced an increase of the inhibitory response which was reversed by naloxone. 6-Hydroxydopamine treatment also resulted in an increase in opioid peptide release. These findings would indicate that the interruption of dopaminergic transmission in the myenteric plexus produces an increase in the release of opioid peptides and suggest an inhibitory modulation of opioid peptidergic neurons by dopamine systems at this level.     

Interaction of ethanol and L-glutamate in the guinea pig ileum myenteric plexus.

The guinea pig ileum longitudinal muscle myenteric plexus has recently been shown to contain receptors for excitatory amino acids like L-glutamate which are pharmacologically similar to the N-methyl-D-aspartate (NMDA) receptor subtype in the central nervous system (CNS). The present study utilized the longitudinal muscle myenteric plexus preparation to determine whether the reported ability of acute ethanol treatment to inhibit NMDA receptor activation in mammalian CNS preparations also occurs in the periphery. In the absence of Mg2+, L-glutamate (3-100 microM) induced transient contractions in longitudinal muscle myenteric plexus that could be blocked by atropine. Contractile responses to L-glutamate were completely blocked by D,L-2-amino-5-phosphonovalerate (APV; 100 microM) and Mg2+ (600 microM). Preincubation with ethanol (30-100 mM) for 2 min inhibited contractions to L-glutamate by up to 50% and caused additive inhibition with 100 microM Mg2+. Ethanol (65 mM) inhibition of L-glutamate (60 microM) contractions increased from 30% after a 2 min preincubation to a maximum of 60% following 10 min. Ethanol (65 mM) inhibited contractions induced by acetylcholine (0.1 microM), 5-hydroxytryptamine (0.1 microM) or histamine (0.3 microM), by no more than 10% suggesting that impairment of smooth muscle or cholinergic neuronal activity were not likely responsible for the 40% inhibition of L-glutamate contractions seen with ethanol. A previously identified contractile response to ethanol (10-300 mM), occurring immediately after addition to the longitudinal muscle myenteric plexus preparation, was still present in Mg2+ deficient buffer.(ABSTRACT TRUNCATED AT 250 WORDS)     

An inhibitory action of tetracyclines on guinea-pig myenteric plexus

In plexus containing preparations of the longitudinal muscle of the guinea-pig ileum, an inhibitory action of tetracyclines on twitch-responses to electrical field stimulation was found. Tetracycline, chlortetracycline, minocycline and doxycycline, but not oxytetracycline (0.02 to 1.6 mmol/l) caused a concentration-dependent presynaptic inhibition of acetylcholine release. The inhibitory effect of the tetracyclines was also obtained after ganglion block by hexamethonium (30 mumol/l). The inhibitory effect of the tetracyclines was not antagonized by piperoxan (2 mumol/l) or yohimbine (1 mumol/l) and was partly reduced by the presence of naloxone (1 to 50 nmol/l). After exposing the preparation the peptidase inhibitors, i.e., to the combination of bestatin (10 mumol/l), captopril (10 mumol/l) and thiorphan (0.3 mumol/l), the inhibitory effect of tetracyclines was significantly increased. From these results it would appear that twitch-inhibition caused by tetracycline, chlortetracycline, minocycline and doxycycline is mainly mediated via the release of endogenous opioids from the myenteric plexus.     

Influence of temperature on the effects of mu-, delta- and kappa-opioid receptor agonists in the guinea-pig ileum myenteric plexus.

Electrically stimulated guinea-pig ileum myenteric plexus-longitudinal muscle was used to determine if changes in temperature alter the inhibitory effects of DAGO ([D-Ala2,N-MePhe4,Gly5-ol]enkephalin, mu-agonist), DPDPE ([D-Pen2,-Pen5] enkephalin, delta-agonist) and U-50,488H (trans-3,4-dichloro)-N-methyl-N-[2-(1- pyrrolidynyl)cyclohexyl]benzeneacetamide methane sulfonate, kappa-agonist). The potency (expressed as the concentration which produces 50% inhibition, IC50) of DAGO and DPDPE was significantly (P < 0.05) decreased at 30 degrees C (8.8 +/- 2.7 x 10(-9) and 8325.2 +/- 1070 x 10(-9) M), when compared to the potency at 37 degrees C (3.8 +/- 0.3 x 10(-9) and 6298.6 +/- 320 x 10(-9) M). Higher temperature (40 degrees C) did not modify the potency of DAGO or DPDPE compared to that at 37 degrees C. However, the potency of U-50,488H was significantly (P < 0.01) increased at 40 degrees C (0.7 +/- 0.0 x 10(-9) M) versus 37 degrees C (2.4 +/- 0.9 x 10(-9) M) or 30 degrees C (2.5 +/- 0.3 x 10(-9) M). The kappa-agonist was more potent than DAGO or DPDPE at 30 or 40 degrees C. These data demonstrate that changes in temperature can alter the potency of opioid agonists.     

The effects of calcium concentration on the inhibition of cholinergic neurotransmission in the myenteric plexus of guinea-pig ileum by adenine nucleotides.

1 Adenosine and the adenine nucleotides AMP, ADP, ATP, cyclic AMP, NAD, NADP and NADH produced a dose-related inhibition of the contractile response of guinea-pig ileum longitudinal muscle-myenteric plexus strips to low frequencies (less than 1 Hz) of electrical field stimulation. 2 These compounds inhibited hexamethonium-sensitive contractions induced by nicotine but did not alter the responses to exogenous acetylcholine, and the acetylcholine output from the myenteric plexus was inhibited by the adenyl compounds. These findings indicate that adenine derivatives act at a presynaptic site on postganglionic cholinergic neurones. 3 The degree of inhibition produced by adenine compounds was inversely related to the calcium concentration of the bath fluid over a range of calcium concentrations (1 to 5 mM) that had no effect on the responses of the muscle to exogenous acetylcholine. 4 The inhibition produced by adenine derivatives was antagonized by theophylline and augmented by dipyridamole. Both of these interactions were sensitive to, and synergistic with, alterations of the concentration of calcium in the bath fluid. 5 The results suggest that adenine compounds inhibit acetylcholine release from the myenteric plexus by diminishing the availability of intracellular calcium ions required for neurotransmitter release.     

ATP and nitric oxide: inhibitory NANC neurotransmitters in the longitudinal muscle-myenteric plexus preparation of the rat ileum.

1. The nature of neurotransmitter(s) involved in non-adrenergic non-cholinergic (NANC) relaxations induced by electrical stimulation (10 s trains, 1-8 Hz) was investigated in the precontracted longitudinal muscle-myenteric plexus preparation of the rat ileum. 2. Electrical stimulation of the tissue induced complex responses, consisting of a primary contraction, a primary relaxation, an off-relaxation and a rebound contraction, which were all tetrodotoxin(TTX)-sensitive. 3. Vasoactive intestinal polypeptide (VIP) and carbon monoxide (CO) did not induce relaxations. alpha-Chymotrypsin did not reduce the relaxations induced by electrical stimulation, while zinc protoporphyrin IX had non-specific effects. 4. Nitric oxide (NO) induced concentration-dependent relaxations. NG-nitro-L-arginine methylester (L-NAME) abolished the primary contractions and off-relaxations, while it partially reduced the primary relaxations. 5. ATP induced relaxations and ATP-desensitization of the tissues partially reduced the primary relaxations. Suramin and reactive blue 2 did not consistently influence the primary relaxations. 6. The ATP-induced relaxations were not influenced by L-NAME or TTX. The inhibitory effect of ATP-desensitization and L-NAME did not summate. 7. The cyclic AMP content of the tissue did not increase upon electrical stimulation or after addition of NO or ATP. The cyclic GMP content of the tissue increased upon electrical stimulation and addition of NO, but not after addition of ATP. 8. It is concluded that the relaxation induced by electrical stimulation consists of two types of responses. The off-relaxation is completely nitrergic, while the primary relaxation is mediated by NO, ATP and an as yet unknown transmitter which is not VIP or CO.     

Morphine tolerance and nonspecific subsensitivity of the longitudinal muscle myenteric plexus preparation of the guinea-pig to inhibitory agonists

1. The sensitivity of the longitudinal smooth muscle/myenteric plexus (LM/MP) to agonists which reduce the amplitude of neurogenic contractions was studied in preparations obtained from animals implanted with either placebo or morphine (75 mg/pellet) pellets 7 days prior. 2. Tolerance or subsensitivity to morphine was observed following chronic treatment with morphine and was revealed as a rightward shift of the concentration-response curve to morphine. The degree of tolerance decayed modestly with time after removal from a morphine containing environment suggesting a time dependence for the loss of subsensitivity to morphine. 3. LM/MP preparations from animals pretreated with morphine also developed subsensitivity to the inhibitory effects of the purine analogue, 2-chloroadenosine. Subsensitivity to 2-chloroadenosine was seen as a parallel rightward shift of the concentration-response curve in morphine-tolerant preparations. The magnitude of the loss in sensitivity was comparable to that observed to morphine. 4. A reduction in sensitivity of the LM/MP following chronic treatment with morphine was also observed to the inhibitory effects of the alpha2 adrenoceptor agonists, clonidine and xylazine. In contrast to the results obtained with morphine and 2-chloroadenosine, the development of subsensitivity to alpha2 adrenoceptor agonists was characterized by a marked reduction in slope and a depression of the maximum response. 5. These data suggest that myenteric neurons possess spare receptors for morphine and 2-chloroadenosine but not for clonidine and xylazine. Furthermore, the studies support the idea that tolerance is associated with a general cellular change or adaptation which impacts on all of these inhibitory substances in such a way as to reduce their efficacy.     

Acetylcholine release from the myenteric plexus of guinea-pig ileum by prostaglandin E1

Release of acetylcholine (ACh) by prostaglandin E₁ from the nerve terminals of the guinea-pig longitudinal muscle strip was studied in order to reveal the effect of PGE₁ on myenteric plexus activity. The ACh released was collected in the presence of physostigmine (2·1 μg ml^?1) and choline (0·1 μg ml^?1) at 38° C. Five to 100 ng ml^?1 PGE₁ enhanced the release dose-dependently. The effect was maintained during the presence of PGE₁ in the organ bath, while rapid tachyphylaxis was observed with the ACh-releasing action of nicotine. Tetrodotoxin or morphine almost completely inhibited the effect of PGE₁ on ACh release. Hexamethonium, in a concentration which completely blocked the effect of nicotine, partially inhibited the effect of PGE₁. In the late phase of nicotine action, the tissue was still sensitive to PGE₁ despite the continued exposure to nicotine. These data suggest the presence in the myenteric plexus of PG receptors which can increase ACh release.     

Localization of endothelin ETB receptors on the myenteric plexus of guinea-pig ileum and the receptor-mediated release of acetylcholine.

1. The type of endothelin (ET) receptor located on the myenteric neurones of guinea-pig ileum was determined by receptor autoradiography and function of the receptor was examined by release experiments of acetylcholine (ACh) from the longitudinal muscle myenteric plexus (LM-MP) preparations. 2. Specific [125I]-ET-1 binding sites were distributed in muscle layers, myenteric and submucous plexuses, and mucosa layers. High-grain densities were detected in both myenteric and submucous plexuses. 3. Binding in the myenteric plexus was abolished by incubation with either IRL 1620 (endothelin ETB receptor agonist) or BQ 788 (endothelin ETB receptor antagonist), but not with BQ 123 (endothelin ETA receptor antagonist). The [125I]-IRL 1620 binding sites were evident in the myenteric plexus. Thus, the endothelin receptor located on the myenteric neurones is of the ETB type. 4. ET-1 (10(-10)-3 x 10(-8) M) and ET-3 (10(-10)-3 x 10(-8) M) evoked 3H outflow from LM-MP preparations of ileum preloaded with [3H]-choline, in a concentration-dependent manner. There was no significant difference between maximum amounts of ET-1-evoked and ET-3-evoked 3H outflow. 5. ET-1 and ET-3 evoked outflow of 3H was BQ 788-sensitive, but BQ 123-insensitive. Both evoked outflows of 3H were Ca(2+)-dependent and tetrodotoxin-sensitive. 6. These results indicate that the endothelin ETB receptor is located on the enteric cholinergic neurones and that stimulation evokes the release of ACh.     

Impulse transmission in the myenteric plexuslongitudinal muscle preparation of the guinea-pig ileum

1. In a preparation consisting of the myenteric plexus and the longitudinal muscle layer removed from a segment of guinea-pig ileum, spontaneous action potentials occurred which were unaffected by tetrodotoxin but suppressed by Mn(2+) and were therefore myogenic.2. A single current pulse of 0.1 ms duration evoked a response consisting of an early action potential followed after a delay of about 200 ms by a complex of biphasic spikes. The first action potential was conducted for no more than 15 mm and the second complex for 30-70 mm.3. Since the first action potential was unaffected by hyoscine or Mn(2+) but abolished by tetrodotoxin, it was due to excitation of nerve fibres. The later complex of spikes was suppressed by hyoscine and Mn(2+) and therefore due to excitation of smooth muscle. It was also inhibited by adrenaline or morphine, compounds which depress acetylcholine release. The evoked smooth muscle response was followed by absence of spontaneous electrical activity for 2-4 seconds.4. The nerves travelling in a longitudinal direction had a mean maximum conduction velocity of 0.65 m/s, an absolute refractory period of 2.8 ms and a relative refractory period of about 20 ms.5. The conduction velocity of the smooth muscle response evoked by stimulation of the nerve with a single pulse was 0.16 m/second. After a single pulse the muscle was inexcitable for 0.7-1.3 s; the delay of transmission from nerve to muscle was 210 ms. When instead of a single pulse a train of two-five pulses at 20 ms intervals was applied, the size, conduction distance and conduction velocity of the evoked smooth muscle response were increased.     

Inhibitory effects of opioids in a circular muscle-myenteric plexus preparation of guinea-pig ileum

Summary The actions of opioids were examined in a strip preparation of the external muscle and myenteric plexus of the guinea-pig ileum cut parallel to the circular muscle. Contractions of the circular muscle induced by electrical stimulation of myenteric neurons were depressed in a concentration-dependent manner by the mu agonists, morphine and DAGO, and by the kappa agonist, U-50,488H. The concentrations of morphine, DAGO and U-50,488H which depressed nerve-mediated contractions by 50% (IC₅₀) were 86 nM, 11 nM and 5.0 nM, respectively. The equilibrium dissociation constants (K _D) for naloxone as an antagonist of the inhibitory effects of DAGO and of U5-0,488H were 5.6 nM and 29.4 nM, respectively. In contrast to the potent inhibitory effects of mu and kappa agonists, the delta-selective agonist, d-Pen-l-Pen, produced only weak inhibition of nerve-mediated contractions. Even at a concentration of 3 M, there was less than 50% inhibition, which was not antagonised by the delta receptor antagonist, ICI 174864. The experiments indicate that both mu and kappa opioid receptors are present on the myenteric neurons supplying the circular muscle and that delta receptors are either absent or ineffectively activated. Send offprint requests to S. M. Johnson     

The action of neurotensin on single myenteric neurones.

The action of neurotensin was studied on single myenteric neurones within ganglia of the myenteric plexus isolated from the guinea-pig ileum. Drugs were applied by adding them to the perfusing Krebs solution. Extracellular recording with glass suction electrodes indicated that neurotensin (100 pM-300 nM) caused a dose-dependent excitation of about 50% of myenteric neurones; the remaining neurones were unaffected. This effect persisted in calcium-free solutions. Intracellular recording showed that a similar proportion of Type 1 myenteric neurones were depolarized by neurotensin: this was associated with an increase in membrane resistance. Type 2 cells were either depolarized or hyperpolarized by neurotensin. The depolarization persisted in calcium-free solutions. The hyperpolarization disappeared in calcium-free solutions, suggesting either that the potential change itself is calcium-dependent or that it was due to release by neurotensin of a hyperpolarizing substance.     

Diprenorphine has agonist activity at opioid kappa-receptors in the myenteric plexus of the guinea-pig ileum

The opioid agonist and antagonist activities of diprenorphine have been tested in four in vitro bioassay preparations. Diprenorphine is an antagonist at delta-receptors in the hamster vas deferens, at mu-receptors in the rat vas deferens and at kappa-receptors in the rabbit vas deferens. In the guinea-pig ileum it is an antagonist at mu-receptors and an agonist at kappa-receptors.